The ready availability of pluripotent adult stem cells would have many important therapeutic applications. Our recent results suggest that the hair follicle is a promising source for such stem cells. Our recent discovery that the neural stem cell marker, nestin is expressed in hair-follicle stem cells (Li, L., et al. Nestin expression in hair follicle sheath progenitor cells. Proc. Natl. Acad. Sci. USA 100, 9658-9662, 2003) suggested that hair-follicle stem cells and neural stem cells have common features. Recently, we have demonstrated that the hair follicle gives rise to blood vessels in vivo with the blood vessels apparently originating from the hair-follicle stem cells (Amoh, Y., et al. Nascent blood vessels in the skin arise from nestin-expressing hair follicle cells. Proc. Natl. Acad. Sci. USA, in press). In preliminary experiments, we have demonstrated in vitro that hair-follicle stem cells can be induced to form neurospheres, which in turn form neurons (Amoh, Y., Li, L., and Hoffman, R.M., unpublished results). These results suggest pluripotent hair follicle adult stem cells could have important therapeutic applications, in particular for neurological diseases. This application utilizes transgenic mice with green fluorescent protein (GFP) under the control of the nestin regulatory sequences as the source of labeled hair follicle cells. In the nestin-GFP transgenic mice, nestin-expressing hair-follicle stem cells are located in the permanent upper hair follicle right below the sebaceous glands and above the errector pili muscle in the follicle bulge area. This group of nestin-expressing cells in the bulge area of the follicle can be activated by anagen induction and form the new hair follicles as well as a blood-vessel network. Immunohistochemical staining revealed that the nestin-GFP-expressing cells in the hair follicle bulge co-express nestin, GFP, and keratin-15 one of the markers of hair-follicle stem cells. The blood vessels formed from the follicle co-express CD-31 an endothelial cell marker as well as GFP. We have shown that the isolated hair-follicle nestin-expressing cells can form neurospheres in vitro that can be switched to form either neurons or blood vessels. The specific aims of this application are as follows: (1) Optimization of isolation of nestin-GFP-expressing hair-follicle stem cells for neurosphere conversion; (2) Determination of the optimal conditions of hairfollicle- stem-cell-derived neurospheres to form neurons. In Phase II, we will use optimized hair follicle derived neorospheres developed in Phase I to determine their therapeutic efficiency to produce neurons, blood vessels, and hair follicles in vivo. Human hair-follicle stem cells will also be developed for therapeutic potential in Phase II. [unreadable] [unreadable]